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ToxinB_ELISA.md

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TcdB ELISA Protocol

Materials and Equipment

tgcBIOMICS Kit for C. difficile Toxin A OR Toxin B

  • ELISA Plate coated with anti-toxin A and anti-toxin B antibodies (ready to use)
  • Dilution buffer (ready to use)
  • Standard Control A (80ng/mL) (ready to use)
  • Standard Control B (80ng/mL) (ready to use)
  • Conjugate 1: anti-toxin A-HRP (ready to use)
  • Conjugate 2: anti-toxin B-HRP (ready to use)
  • 10X Wash Buffer (to be diluted)
  • TMB - substrate (ready to use)
  • Stop Reagent (ready to use)

Protocol

  • Weigh out 30-50 mg of fecal samples (ideally the close the weight of the samples to each other, the better) in a eppendorf tube. The samples can be weighed out prior to the procedure and store at -80C.
  • Take fecal samples and reagent kit out to let them return to room temperature.
  • Transfer 450uL of Dilution Buffer to each tube and use the pipette tip to break up the fecal pellets and mix with the dilution buffer.
  • Vortex to further mix the fecal sample and dilution buffer. Let samples sit on the tube rack to have the particulates settle.
  • When we wait, we can prepare the standards. Serially dilute the 80ng/mL stock to 40ng/mL, 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0.63ng/mL, and 0.31ng/mL. This can be done by diluting 110uL of the standard in 110uL of Dilution Buffer.
  • Carefully transfer 100uL of the liquid layer of the samples and the standards to the plate. The blank can just be 100uL of Dilution Buffer. Record the well ID.
  • Seal the plate with the sticky aluminum seal to prevent evaporation. Incubate the plate for 60min at 37C.
  • Dilute 10X Wash Buffer by adding 1 part of the dilution buffer to 9 parts of mqH2O.
  • Dilute the conjugate by adding 1 part of the conjugate to 2 parts of Dilution Buffer.
  • Pipette out the content in each well and dispense into a waste beaker (to prevent cross-contamination) and wash each well with 300uL of 1X Wash Buffer three times.
  • Dilute the conjugate with 2 parts of dilution buffer. (There’s total of 3.5 mL of conjugate in the bottle so be stingy on this part and use the just-enough volume because there’s no surplus in the bottle.
  • Add 100uL of the diluted conjugate into each well and seal the plate. Incubate the place for 30 minutes at 37C.
  • Pipette out the content in each well and dispense into a waste beaker (to prevent cross-contamination) and wash each well with 300uL of 1X Wash Buffer three times.
  • Dry the plate by striking the plate onto a dry paper towel.
  • Add 100uL of the substrate to each well and incubate the plate at room temperature for 15 minutes. It’s better to have the plate covered and shielded from light.
  • Add 50uL of the stop reagent to each well to stop the reaction and color development.
  • Measure OD at 450nm and 620nm and use the difference in OD between the two wavelengths to plot.

Note: The result for the final estimated concentration of the toxin depends on couple of variables, including incubation time and temperature and etc. Given that minor variations are possible, always run the standard together with the samples for each assay.